The Effects Of Down-regulated CrkL On Biological Behavior Of Mouse Hepatocarcinoma Cell Hca-P

Background: The early lymph node metastasis (LNM) of malignant tumor couldinduce high mortality and poor prognosis, which the uncertain mechanisms are alwaysdifficult problem in the field of oncology. Mouse hepatocarcinoma cell Hca-F withhigh metastatic potential (75%) and Hca-P with low metastatic potential (25%) are apair of synogenetic hepatocellular carcinoma ascites cell lines established, those twocell lines sharing same genetic background are ideal experimental subjects for thestudy of LNM.CT10regulator of kinase (Crk) was originally identified as an oncogene productof v-Crk encoded in a chicken tumor retrovirus, CT10, that comprised Src homology2(SH2) and SH3domains. The cellular homologues CrkI and CrkII, and the relatedCrk-like (CrkL) are ubiquitously expressed and conserved across eukaryotic organ–isms. CrkL (Crk-Llike) is a member of human Crk adaptor protein family,accumulated evidence indicates that the abnormal expression of CrkL plays animportant role aggressive and malignant behaviors of human cancers, however, therelationship between CrkL and hepatocellular carcinoma LNM rarely reported.Our previous studies found that CrkL differentially expressed in Hca-F and Hca-Pcell lines, the protein expression level of CrkL in Hca-P is3.05-fold higher than thatof CrkL in Hca-F cells (P<0.05), indicating the expression level of CrkL is closelyrelated with tumor lymphatic metastasis, and might be a potential therapeutic target in the treatment of hepatocellular carcinoma lymphatic metastatic. In the present study,we down-regulate the protein expression of CrkL in Hca-P cell line by RNAinterfering method to further investigate the effect of CrkL on cell malignantbehaviors of tumor lymphatic metastasis.Objective:1. To construct the expression vectors pGPU6/GFP/Neo-shRNA-CrkL andobtain the monoclonal pGPU6/GFP/Neo-shRNA-CrkL-Hca-P cell with CrkL stablyknockdown;2. To investigate the effect of down-regulated CrkL on Hca-P cellproliferation, migration, invasion ability in vitro;3. To investigate the effect ofdown-regulated CrkL on tumor formation and lymph node metastasis of Hca-Ptumor-bearing mice in vivo.Methods:1.The siRNAs targeting the CrkL were designed according to the mRNAsequence of CrkL (GenBank: BC131984) using siDirect and whitehead software,meanwhile, one with non-targeting sequence to be used as a negative control. Thenthe pGPU6/GFP/Neo-shRNA-CrkL and pGPU6/GFP/Neo-shRNA-control expressionvectors were stably transfected into Hca-P by Lipofectamine2000method, themonoclonal pGPU6/GFP/Neo-shRNA-CrkL cell with CrkL stably knockdown andpGPU6/GFP/Neo-shRNA-control-Hca-P cell were obtained by G418selection andlimited dilution, and the protein expression of CrkL in monoclonal cell, negativecontrol cell and Hca-P cell were confirmed by western blot;2. CCK-8(cell countingkit-8) measured the influence of CrkL down-trgulation on Hca-P cell proliferationability. Transwell chamber was used to detect the influence of CrkL down-trgulationon Hca-P cell migration and invasion ability.3. The mice foot-pad planting methodanalyzed the effect of CrkL down-regulation on tumor formation and LNM of Hca-Ptumor-bearing mice in vivo.Results:1. We designed three siRNA of CrkL and successfully constructed3recombinant plasmid including pGPU6/GFP/Neo-shRNA-CrkL-707,pGPU6/GFP/Neo-shRNA-CrkL-732and pGPU6/GFP/Neo-shRNA-CrkL-560, andalso screened15monoclonal cell lines with CrkL stably knockdown. The results ofwestern blot indicated that the protein level of CrkL in707-7、732-3and560-4monoclonal cell lines significantly down-regulated63.34%,93.12%and96.15% respectively compared to pGPU6/GFP/Neo-shRNA-control and Hca-P cell lines,suggesting these cell lines could be used as research object for subsequent experimentof cell malignant activity.2. CCK-8assay showed that the monoclonal cell lines707-7、732-3and560-4grew faster than the pGPU6/GFP/Neo-shRNA-control after96h (P<0.05); Transwell chamber assay showed that the number of monoclonal celllins cells migrated and invaded through the filter were more than that of negativecontrol pGPU6/GFP/Neo-shRNA-control (P<0.05);3. In vivo animal experimentresults shown that CrkL down-regulation promote the tumor formation and LNM ofmice foot-pad.Conclusion:1. We successfully constructed pGPU6/GFP/Neo-shRNA-CrkLexpression vector and screened15monoclonal cells with CrkL down-regulated stablyand different interference efficiency, which provide a solid base for further studyingthe functions of CrkL in hepatoma lymphatic metastasis;2. The down-regulation ofCrkL could enhance proliferation, migration and invasion potential of Hca-P cell;3.The down-regulation of CrkL could promote tumor formation and LNM rate of micefoot-pad;4. CrkL play a crucial role in hepatocarcinoma carcinoma cell malignantbehavior and has potential for use as a novel biomaker for therapy ofhepatocarcinoma carcinoma.